The purpose of the proposed research is to characterize and to determine the physiological significance of a phosphorylation system (endogenous protein kinase and substrate) detected in the outer plasma membrane of 3T3 cells. The activity is correlated with cell growth in 3T3 cells: it is greater in growing cells than in quiescent ones; it decreases as cell density increases; it is 5-10 fold greater in SV40-3T3 cells than in normal 3T3. Stimulation of quiescent cells with serum brings about a 2-4 fold increase in incorporation of phosphate as does also treatment with small amounts of insulin, prostaglandin E1 or F2alpha. The increase in phosphorylation appears to be a G1 event. Inhibitors of RNA or protein synthesis block the increase in phosphorylation as well as passage through G1. Hydroxyurea, which blocks at the G1/S boundary, does not prevent the increase in phosphorylation. We plan to characterize the protein kinases and substrates in quiescent and growing 3T3 cells, in temperature-sensitive cell cycle mutants, and in several classes of SV40 transformed 3T3 cells. We will look for quantitative and qualitative changes in cellular phosphorylation. We will assay incorporation of 32P-phosphate from gamma32P-ATP into acid-precipitable protein of intact cells and isolated membrane preparations. We will also examine radioautographic patterns of SDS polyacrylamide gel electrophorograms of phosphorylated cells. Membrane preparations from normal and transformed cells will be phosphorylated to try to determine the sidedness of the enzymes and substrates. Immunoprecipitation as well as selective membrane extraction procedures will be used to begin examining substrates and enzymes independently.